The long-term objective of this research is to understand the molecular mechanism of the voltage, Ca2+, and Mg2+ dependent activation of large-conductance K+ channels (BK channels). BK channels have road physiological functions, including the modulation of neurotransmitter release and the control of blood vessel diameters. As a consequence of these physiological functions BK channels are of significant clinical importance. For example, abnormal activity of BK channels has been associated with hypertension in animal models; their increased activity may reduce the incidence of ischemia- reperfusion-induced cardiac arrhythmia. In the activation of BK channels voltage induces movements of the voltage sensor in the channel, Ca2+ or Mg2+ binds to the channel to cause conformational changes in the channel protein to open he activation gate. Now the structure of the K+ channel pore has been solved; protein sequences underlying he activation gate, the voltage sensor, and the Ca2+ binding site have been identified. However, the manner n which voltage sensor movements, Ca2+ or Mg2+ binding are coupled to the opening of the activation gate remains unknown. Until the structural and energetic basis of these couplings is elucidated, how voltage, Ca2+ and Mg2+ sensitivities are modulated in various BK channels to subserve their physiological functions cannot be understood. Based on previous studies, we hypothesize that a structural domain of the channel protein that is physically close to the activation gate (the RCK domain for Regulating the conductance of K+ channels) is central in these couplings. Recently, the X-ray crystal structure of the RCK domain has been solved. Guided by the structural data we will perturb the channel structure using molecular biology and determine its impact on the energetic contribution of Ca2+, Mg2+, or voltage to channel opening using our recently developed electrophysiological approaches. We will also use approaches of protein biochemistry and nuclear magnetic resonance spectroscopy (NMR) to map specific intramolecular protein interactions that nay be altered during channel activation and hence control channel function. These experiments will provide a foundation for understanding how various BK channels play their role in physiological processes and define targets on BK channels for therapeutic purposes. They will also contribute to our understanding of ion channel gating in general.